An automated sample loading system for cell sorting

Abstract

This invention discloses an automated sample loading system for cell separation, comprising: a pump device, a reagent switching device, a sample container, a first solenoid valve, a second solenoid valve, a third solenoid valve, a weighing sensor, a bubble sensor, a controller, and a microfluidic chip. Sample loading is achieved through the pump device, reagent switching device, microfluidic chip, rare cell tube, sample tube, and sheath fluid tube, eliminating the need for pipettes and significantly reducing consumable usage. The sample loading process does not require frequent pipette tip changes; only the reagent switching device needs to be controlled. The response time is reduced from fractions of a second to milliseconds, improving efficiency. Furthermore, no space is required for pipetting routes or pipette tip boxes, resulting in a compact structure that facilitates device miniaturization and placement.

Description

Technical Field

[0001] This invention relates to the field of cell sorting technology, and more specifically, to an automated sample loading system for cell sorting. Background Technology

[0002] Rare cell sorting is a technology that precisely "retrieves" minute quantities of target cells from a massive number of cells. It enables scientific research and clinical medicine to directly study those difficult-to-obtain but crucial cells or subpopulations, leading to breakthroughs in understanding life mechanisms and disease diagnosis and treatment. At the research level, this technology can be used to reveal cellular heterogeneity and discover novel cell types; at the clinical level, it can be used for early diagnosis and screening of tumor cells, real-time monitoring of treatment efficacy and prognostic assessment, and guiding precision medicine.

[0003] Sample loading systems enable rapid and efficient sorting of rare cells. Currently, most cell sorting systems on the market utilize pipetting workstations for sample loading. A pipetting workstation is a device that is programmed to automatically perform a series of operations, including sample dispensing, reagent addition, and liquid transfer, using pipettes. While pipetting workstations automate sample loading, greatly improving the convenience of cell sorting, they also have some drawbacks: 1. High consumption of consumables: Since the pipetting process relies on pipettes, the pipette tip needs to be replaced every time a reagent is added, resulting in a high consumption of consumables and a large amount of waste.

[0004] 2. Inefficient: The sample loading process requires frequent changes of the nozzle, which is not only cumbersome to operate and difficult to plan the route, but also very time-consuming.

[0005] 3. Difficulty in miniaturizing the equipment: Sufficient space needs to be reserved in the equipment to place the pipette tip and set up the pipetting path, which is not conducive to the realization of compactness and miniaturization of the equipment.

[0006] It is evident that existing sample loading systems suffer from drawbacks such as high consumption of consumables, low efficiency, and difficulty in miniaturization. Summary of the Invention

[0007] This invention provides an automated sample loading system for cell sorting, which can reduce the amount of consumables used, improve efficiency, and has a compact structure that is easy to miniaturize. The specific technical solution is as follows.

[0008] In a first aspect, the present invention provides an automated sample loading system for cell separation, comprising: a pump device, a reagent switching device, a sample container, a first solenoid valve, a second solenoid valve, a third solenoid valve, a weighing sensor, a bubble sensor, a controller, and a microfluidic chip, wherein the pump device comprises an industrial injection pump, a valve head, and a sample injector. The industrial injection pump is connected to the injector. The injector, cell collection chamber, waste liquid pool and the sample outlet of the microfluidic chip are all connected to the valve head. The pipeline between the sample outlet and the valve head is equipped with the bubble sensor. When the bubble sensor detects a bubble, it sends an early warning message to the controller. The first inlet of the microfluidic chip is connected to the common tube of the reagent switching device through a sample tube, the second inlet of the microfluidic chip is connected to the common tube through a sheath fluid tube, the sample container is connected to the sample tube through a rare cell tube, the sample container contains a sample of rare cells with magnetic beads attached to its surface, the weighing sensor is connected to the sample container, the first solenoid valve is located in the rare cell tube, the second solenoid valve is located in the sample tube near the common tube, and the third solenoid valve is located in the sheath fluid tube near the common tube; The controller controls the second and third solenoid valves to open, controls the valve head to connect with the sample outlet, and controls the common tube to connect sequentially with the reagent storage tanks corresponding to the pretreatment reagents and culture media in the reagent switching device in a first order. Simultaneously with the common tube connecting with the first reagent storage tank, the controller starts the industrial injection pump. When the common tube connects with the reagent storage tank corresponding to the culture media, the controller closes the second solenoid valve and opens the first solenoid valve. The industrial injection pump draws the sample into the microfluidic chip for cell sorting. When the sample is emptied based on the weight sent by the weighing sensor, the controller switches the valve head to connect with the waste liquid tank. After the waste liquid is emptied, the controller switches the valve head to connect with the sample outlet. After the sorted suspension enters the injector, the controller switches the valve head to connect with the cell collection chamber, and the suspension is drawn into the cell collection chamber.

[0009] Optionally, the reagent switching device includes a multi-channel switching valve, a reagent storage tank with the same number of channels as the multi-channel switching valve, and multiple liquid level sensors respectively disposed in each reagent storage tank; Each liquid level sensor detects the liquid level of the reagent in its corresponding reagent storage tank and sends the data to the controller; When the controller determines that all liquid levels are not lower than the preset liquid level threshold, it controls the second solenoid valve and the third solenoid valve to open.

[0010] Optionally, the controller outputs an alarm message when it determines that there is a liquid level below a preset liquid level threshold.

[0011] Optionally, the controller determines that the weight of the sample has not changed within a preset time period based on the weight sent by the weighing sensor, controls the third solenoid valve to close, controls the valve head to switch to connect with the waste liquid pool, controls the valve head to switch to connect with the sample outlet after the waste liquid in the sample injector is emptied, controls the valve head to switch to connect with the sample outlet after the sorted suspension enters the sample injector, controls the valve head to switch to connect with the cell collection chamber, and the suspension is drawn into the cell collection chamber.

[0012] Optionally, after sorting is completed, the controller controls the first solenoid valve to close, controls the second solenoid valve to open, and controls the common tube to connect sequentially with the reagent storage tanks corresponding to the multiple reagents for staining in the reagent switching device in a second order. Each reagent for staining flows sequentially into the microfluidic chip through the sample tube to stain the sorted suspension.

[0013] Optionally, the sample container is connected to the sample tube near the first inlet via the rare cell tube.

[0014] Optionally, the microfluidic chip includes a sample layer, a functional layer, and a magnetic field source; The sample layer includes a flow channel layer and a sealing membrane. The flow channel layer is provided with a first inlet, a second inlet and the sample outlet, and has flow channels processed inside. The sealing membrane is attached to the open surface of the flow channel layer to seal the flow channels inside the flow channel layer. The functional layer is located below the sample layer. The functional layer includes a patterned substrate, wherein a plurality of recessed slots are sequentially formed along the length direction of the patterned substrate. An amorphous alloy soft magnetic tape is embedded in each recessed slot, and the upper surface of the amorphous alloy soft magnetic tape is flush with the upper surface of the patterned substrate. The amorphous alloy soft magnetic tape faces the sealing film. The amorphous alloy soft magnetic tape is arranged sequentially along the length direction of the patterned substrate to form an array distribution parallel to the extension direction of the flow channel of the sample layer. The magnetic field source is located at the end where the sample outlet is located and on the side directly opposite the functional layer, so that a gradient magnetic field that gradually increases from the inlet to the sample outlet is formed in the flow channel for cell sorting.

[0015] Optionally, the above-mentioned automated sample loading system for cell separation also includes a support, wherein the loading surface of the weighing sensor is fixedly connected to the support, and the sample container is fixed to the support.

[0016] Optionally, the liquid level sensor is a capacitive liquid level sensor.

[0017] Optionally, the valve head is a T-type three-way valve head.

[0018] As described above, the automated sample loading system for cell separation provided in this embodiment of the invention includes: a pumping device, a reagent switching device, a sample container, a first solenoid valve, a second solenoid valve, a third solenoid valve, a weighing sensor, a bubble sensor, a controller, and a microfluidic chip. The pumping device includes an industrial syringe pump, a valve head, and a syringe injector. The industrial syringe pump is connected to the syringe injector. The syringe injector, cell collection chamber, waste liquid pool, and the sample outlet of the microfluidic chip are all connected to the valve head. A bubble sensor is installed on the pipeline between the sample outlet and the valve head. When the bubble sensor detects a bubble, it sends an early warning message to the controller. The first inlet of the microfluidic chip is connected to the common tube of the reagent switching device through the sample tube. The second inlet of the microfluidic chip is connected to the common tube through the sheath fluid tube. The sample container is connected to the sample tube through a rare cell tube. The sample container contains a sample of rare cells with magnetic beads attached to its surface. The weighing sensor is connected to the sample container. A first solenoid valve is located in the rare cell tube, a second solenoid valve is located in the sample tube near the common tube, and a third solenoid valve is located in the sheath fluid tube near the common tube. The controller controls the second and third solenoid valves to open, connects the valve heads to the sample outlet, and controls the common tube to connect sequentially to the reagent storage tanks corresponding to multiple pretreatment reagents in the reagent switching device in a first order. Simultaneously, when the common tube connects to the first reagent storage tank, the controller starts the industrial injection pump. When the common tube connects to the reagent storage tank corresponding to the culture medium, the controller closes the second solenoid valve and opens the first solenoid valve. The industrial injection pump draws the sample into the microfluidic chip for cell sorting. When the sample is emptied based on the weight sent by the weighing sensor, the controller switches the valve head to connect to the waste liquid tank. After the waste liquid is emptied, the controller switches the valve head to connect to the sample outlet. After the sorted suspension enters the injector, the controller switches the valve head to connect to the cell collection chamber, and the suspension is drawn into the cell collection chamber. Therefore, sample loading is achieved through a pump device, reagent switching device, microfluidic chip, rare cell tube, sample tube and sheath tube, eliminating the need for pipettes, greatly reducing the amount of consumables used. The sample loading process also does not require frequent replacement of pipette tips, only the reagent switching device needs to be controlled. The response time is shortened from seconds to milliseconds, improving efficiency. Furthermore, there is no need to reserve space for pipetting routes and pipette tip boxes, and the compact structure makes it easy to miniaturize the equipment and place it conveniently.

[0019] The innovative aspects of this invention include: 1. Sample loading is achieved through a pump device, reagent switching device, microfluidic chip, rare cell tube, sample tube, and sheath fluid tube, eliminating the need for pipettes and greatly reducing consumable usage. The sample loading process also eliminates the need for frequent pipette tip replacements, requiring only control of the reagent switching device. The response time is reduced from seconds to milliseconds, improving efficiency. Furthermore, there is no need to reserve space for pipetting routes or pipette tip boxes, resulting in a compact structure that facilitates device miniaturization and convenient placement.

[0020] 2. Cell loss can be reduced by using a T-type three-way valve head.

[0021] 3. The sample container is connected to the sample tube near the first inlet via a rare cell tube, thereby maximizing its proximity to the first inlet. This reduces non-specific adsorption of rare cells as they flow through the tube accessories, ensuring the sensitivity of cell sorting.

[0022] 4. By setting up a support frame, the sample in the sample container can be weighed using a weighing sensor.

[0023] 5. The liquid level sensor is a capacitive liquid level sensor, which can reduce costs.

[0024] 6. By introducing sheath fluid through the second inlet of the flow channel layer in the sample layer, a stable boundary layer can be formed, ensuring the flow state of the sample in the flow channel. The sealing membrane adheres to the open surface of the flow channel layer to achieve sealing. The patterned substrate in the functional layer has a countersunk groove embedded with amorphous alloy soft magnetic tape material, and the upper surface is flush with the array distribution parallel to the flow channel extension direction. Combined with the local strengthening effect of the amorphous alloy with high relative magnetic permeability on the magnetic field, it can work with the magnetic field source to construct a gradient magnetic field that gradually increases from the inlet to the outlet in the flow channel. This allows rare cells with different numbers of magnetic beads to be captured successively at different positions in the flow channel, thereby achieving cell sorting.

[0025] 7. If the weight of the sample, determined by the weight sent by the weighing sensor, does not change within a preset time period, forced sample loading can be performed by controlling the third solenoid valve to close and the valve head to switch, thereby completing the collection of rare cells.

[0026] 8. After sorting, the first solenoid valve is closed and the second solenoid valve is opened. The common tube is connected to the reagent storage tanks corresponding to the multiple reagents used for staining in the reagent switching device in the second order to stain the sorted suspension, thereby verifying whether the sorted cells are the required rare cells.

[0027] 9. The cell sorting and loading process is fully automated and requires no human intervention.

[0028] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0030] Figure 1A schematic diagram of an automated sample loading system for cell sorting provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of the pumping device provided in an embodiment of the present invention; Figure 3 A schematic diagram of the structure of a microfluidic chip provided for an embodiment of the invention.

[0031] Figures 1-3 The components include: 1. Pump device, 11. Industrial injection pump, 12. Valve head, 13. Injector, 2. Reagent switching device, 21. Multi-channel switching valve, 22. Reagent storage tank, 3. Sample container, 4. First solenoid valve, 5. Second solenoid valve, 6. Third solenoid valve, 7. Microfluidic chip, 71. Sample layer, 711. Flow channel layer, 712. Sealing membrane, 72. Functional layer, 721. Patterned substrate, 722. Amorphous alloy soft magnetic tape, 73. Magnetic field source, 74. First inlet, 75. Second inlet, 76. Sample outlet, 20. Cell collection chamber, 30. Waste liquid pool, 40. Sample tube, 50. Common tube, 60. Sheath fluid tube, 70. Rare cell tube. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0033] It should be noted that the terms "comprising" and "having," and any variations thereof, in the embodiments and drawings of this invention are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or device that includes a series of steps or units is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products, or devices.

[0034] This invention discloses an automated sample loading system for cell sorting, which can reduce the amount of consumables used, improve efficiency, and has a compact structure that is easy to miniaturize. The embodiments of this invention are described in detail below.

[0035] Figure 1 This is a schematic diagram of an automated sample loading system for cell separation provided in an embodiment of the present invention. Figure 2 This is a schematic diagram of the pumping device provided in an embodiment of the present invention. See also: Figure 1 and Figure 2The automated cell fractionation and loading system provided in this embodiment of the invention includes: a pump device 1, a reagent switching device 2, a sample container 3, a first solenoid valve 4, a second solenoid valve 5, a third solenoid valve 6, a weighing sensor, a bubble sensor, a controller, and a microfluidic chip 7. The pump device 1 includes an industrial syringe pump 11, a valve head 12, and a sample injector 13. The first solenoid valve 4, the second solenoid valve 5, and the third solenoid valve 6 are all two-way solenoid valves.

[0036] The industrial syringe pump 11 is connected to the injector 13. The injector 13, cell collection chamber 20, waste liquid reservoir 30, and the sample outlet of the microfluidic chip 7 are all connected to the valve head 12. The valve head 12 can switch between the sample outlet, cell collection chamber 20, and waste liquid reservoir 30. The valve head 12 is a T-type three-way valve head. Using the T-type three-way valve head can reduce cell loss.

[0037] A bubble sensor is installed in the pipeline between the sample outlet and the valve head 12. When the bubble sensor detects a bubble, it sends an early warning message to the controller to indicate that the detection result is abnormal. The detection result is for reference only. The size of the bubble detected by the bubble sensor can be set. The size of the bubble is larger than the preset size because a bubble that is too small will not affect the detection result. For example, the bubble sensor can be set to detect bubbles with a diameter of 100μm.

[0038] The first inlet of the microfluidic chip 7 is connected to the common tube 50 of the reagent switching device 2 through the sample tube 40, the second inlet of the microfluidic chip 7 is connected to the common tube 50 through the sheath fluid tube 60, and the sample container 3 is connected to the sample tube 40 through the rare cell tube 70.

[0039] In one implementation, the sample container 3 is connected to the sample tube 40 near the first inlet via the rare cell tube 70, thereby maximizing its proximity to the first inlet and reducing non-specific adsorption of rare cells as they flow through the tube, thus ensuring the sensitivity of cell sorting.

[0040] The sample container 3 contains a sample of rare cells with magnetic beads attached to its surface. A first solenoid valve 4 is located in the rare cell tube 70, a second solenoid valve 5 is located in the sample tube 40 near the common tube 50, and a third solenoid valve 6 is located in the sheath fluid tube 60 near the common tube 50. A weighing sensor is connected to the sample container 3. Rare cells are cell types that are present in very small amounts in peripheral blood or tissues, typically accounting for less than 0.1%, but have significant biological or clinical importance, such as circulating tumor cells, stem cells, and specific immune cells.

[0041] In one implementation, a support frame is also included, with the load-bearing surface of the weighing sensor fixedly connected to the support frame, and the sample container 3 fixed to the support frame. The sample container 3 can be fixed to the support frame by suspension, in which case the weighing sensor measures the sum of the weight of the support frame, the sample container 3 itself, and the sample inside the sample container 3.

[0042] Therefore, by setting up a support frame, the sample in sample container 3 can be weighed using a weighing sensor.

[0043] Cell sorting using the automated sample loading system provided in this invention can be divided into the following steps: 1. Chip preprocessing The controller opens the second solenoid valve 5 and the third solenoid valve 6, opening the sample channel and the sheath fluid channel. The control valve head 12 connects to the sample outlet, and the control common tube 50 connects sequentially to the reagent storage tanks 22 corresponding to multiple pretreatment reagents and culture media in the reagent switching device 2. Simultaneously with the connection of the common tube 50 to the first reagent storage tank 22, the industrial injection pump 11 starts, sequentially drawing multiple pretreatment reagents into the injector 13. During the process of drawing the pretreatment reagents into the injector 13, each pretreatment reagent flows through the common tube 50, sample tube 40, sheath fluid tube 60, and microfluidic chip 7, forming a protective film on the components it passes through, preparing for subsequent cell sorting.

[0044] The first order can be set based on experience, and the pretreatment reagents may include pure water, ethanol, 84 disinfectant, PBS (Phosphate Buffer Saline), F (Pluronic) 68 and BSA (Bovine Serum).

[0045] See also Figure 1 The reagent switching device 2 includes a multi-channel switching valve 21, a reagent storage tank 22 with the same number of channels as the multi-channel switching valve 21, and a plurality of liquid level sensors respectively disposed on each reagent storage tank 22. The liquid level sensors are disposed on the reagent storage tank 22 by fixing them to the outer periphery of the reagent storage tank 22. For example, the number of reagent storage tanks 22 is 10.

[0046] Before chip preprocessing, status detection is also required. After the automatic sample loading system for cell sorting provided in this embodiment of the invention is powered on, each liquid level sensor detects the liquid level of the corresponding reagent storage tank 22 and sends it to the controller.

[0047] When the controller determines that all liquid levels are not below the preset liquid level threshold, it indicates that there are remaining reagents available for pretreatment. At this point, the controller opens the second solenoid valve 5 and the third solenoid valve 6. The liquid level sensor is a capacitive type, which reduces costs.

[0048] When the controller detects that the liquid level is below the preset threshold, it indicates that there is reagent with an excessively low liquid level. At this time, an alarm message is output so that reagent can be added to ensure the normal progress of subsequent cell sorting.

[0049] 2. Cell sorting After multiple pretreatment reagents are sequentially drawn into the injector 13, the pretreatment is completed. When the common tube 50 is connected to the reagent storage tank 22 corresponding to the culture medium, the second solenoid valve 5 is closed and the first solenoid valve 4 is opened. The industrial injection pump 11 draws the sample into the microfluidic chip 7 for cell sorting.

[0050] Figure 3 A schematic diagram of the structure of the microfluidic chip 7 provided in the embodiment of the invention is shown below. Figure 3 The microfluidic chip 7 includes a sample layer 71, a functional layer 72, and a magnetic field source 73.

[0051] The sample layer 71 includes a flow channel layer 711 and a sealing membrane 712. The flow channel layer 711 is provided with a first inlet 74, a second inlet 75 and a sample outlet 76, and has flow channels processed inside. The sealing membrane 712 is attached to the open surface of the flow channel layer 711 to seal the flow channels inside the flow channel layer 711. The functional layer 72 is located below the sample layer 71. The functional layer 72 includes a patterned substrate 721, wherein a plurality of countersunk grooves are sequentially formed along the length direction of the patterned substrate 721. An amorphous alloy soft magnetic tape 722 is embedded in each countersunk groove, and the upper surface of the amorphous alloy soft magnetic tape 722 is flush with the upper surface of the patterned substrate 721. The amorphous alloy soft magnetic tape 722 faces the sealing film 712. The amorphous alloy soft magnetic tape 722 is arranged sequentially along the length direction of the patterned substrate 721 to form an array distribution parallel to the flow channel extension direction of the sample layer 71. The magnetic field source 73 is located at the end of the sample outlet and is on the side directly opposite the functional layer 72, so that a gradient magnetic field that gradually increases from the inlet to the sample outlet is formed in the flow channel for cell sorting.

[0052] The magnetic field source 73 can be a rectangular permanent magnet with its long side parallel to the flow channel. The flow channel has a laminar flow structure with the sheath fluid on the outside and the sample flow on the inside, which confines the sample flow to the center of the flow channel and prevents cells from drifting away from the magnetic field's area of ​​action.

[0053] The function of the functional layer 72 is to form a local magnetic field that is concentrated on the flow channel region of the sample layer 71 through the high relative magnetic permeability of the amorphous alloy soft magnetic tape 722. Since the magnetic field source 73 is located at the end of the sample outlet 76 and is directly opposite the functional layer 72, the closer it is to the sample outlet end, that is, the closer it is to the magnetic field source 73, the stronger the initial magnetic field generated by the magnetic field source 73 itself is. The strengthening effect of the amorphous alloy at the corresponding position on the magnetic field is also more significant. This forms a gradient magnetic field that gradually increases from the inlet to the sample outlet 76 in the flow channel for cell sorting.

[0054] After the sheath fluid enters through the second inlet 75, it forms a fluid layer along the inner wall of the flow channel, enveloping the sample flow. This fluid layer effectively constrains the diffusion and turbulence of the sample flow, ensuring that the cells in the sample maintain an orderly linear motion within the flow channel, providing a stable fluid environment for subsequent magnetic field sorting. Rare cells with the most magnetic beads bound to their surfaces experience the strongest magnetic force and can overcome fluid friction even in the relatively weak magnetic field at the flow channel inlet, thus being the first to be drawn towards the surface of the sealing membrane 712 for capture.

[0055] Rare cells with fewer magnetic beads are unable to be captured in the weak magnetic field at the inlet due to the smaller magnetic force they experience. They continue to move with the sample flow towards the outlet. As the magnetic field strength in the channel gradually increases, these cells will be subjected to sufficient magnetic force in turn during the process and will eventually be captured at the corresponding position, thus completing cell sorting.

[0056] Therefore, the introduction of sheath fluid through the second inlet 75 of the flow channel layer 711 in the sample layer 71 can form a stable boundary layer, ensuring the flow state of the sample in the flow channel. The sealing membrane 712 fits against the open surface of the flow channel layer 711 to achieve sealing. The patterned substrate 721 in the functional layer 72 has a countersunk groove embedded with an amorphous alloy soft magnetic tape 722, and its upper surface is flush with the array distribution parallel to the flow channel extension direction. Combined with the local strengthening effect of the amorphous alloy with high relative magnetic permeability on the magnetic field, it can work with the magnetic field source 73 to construct a gradient magnetic field that gradually increases from the inlet to the outlet in the flow channel, so that rare cells with different numbers of magnetic beads are captured successively at different positions in the flow channel, thereby achieving cell sorting.

[0057] 3. Cell collection When the sample is emptied based on the weight sent by the weighing sensor, cell sorting is confirmed to be complete. The control valve head 12 is then switched to connect with the waste liquid pool 30 to discharge the waste liquid into the waste liquid pool 30. After the waste liquid is emptied, the control valve head 12 is switched to connect with the sample outlet 76 to draw the sorted suspension into the sampler 13. After the sorted suspension enters the sampler 13, the control valve head 12 is switched to connect with the cell collection chamber 20, and the suspension is drawn into the cell collection chamber 20, thereby completing the collection of rare cells.

[0058] Among them, determining sample emptying based on the weight sent by the weighing sensor can be done by judging whether the weight obtained by subtracting the weight of the support and the weight of the sample container 3 itself from the weight sent by the weighing sensor is 0. If it is 0, the sample is determined to be empty.

[0059] In one implementation, the controller determines that the weight of the sample has not changed within a preset time period based on the weight sent by the weighing sensor. This indicates that there may be blockages in the sample, such as blood clots. In this case, forced sample loading is required. Specifically, the controller closes the third solenoid valve 6, switches the control valve head 12 to connect with the waste liquid pool 30, and after the waste liquid in the injector 13 is emptied, the control valve head 12 switches to connect with the sample outlet 76. After the sorted suspension enters the injector 13, the control valve head 12 switches to connect with the cell collection chamber 20, and the suspension is drawn into the cell collection chamber 20.

[0060] Therefore, when the weight of the sample, determined by the weight sent by the weighing sensor, does not change within a preset time period, forced loading can be performed by controlling the third solenoid valve 6 to close and the valve head 12 to switch, thereby completing the collection of rare cells.

[0061] In one implementation, after sorting, the controller closes the first solenoid valve 4 and opens the second solenoid valve 5. The controller then connects the common pipe 50 sequentially to the reagent storage tanks 22 corresponding to multiple staining reagents in the reagent switching device 2, according to a second sequence. Each staining reagent flows sequentially through the sample tube 40 into the microfluidic chip 7 to stain the sorted suspension. The multiple staining reagents may include a fixative, a permeabilizer, and a staining agent. The second sequence can be set according to staining requirements; for example, the fixative may be Triton, and the permeabilizer may be paraformaldehyde.

[0062] Therefore, after sorting, by controlling the first solenoid valve 4 to close, controlling the second solenoid valve 5 to open, and controlling the common pipe 50 to connect to the reagent storage tanks 22 corresponding to the multiple reagents used for staining in the reagent switching device 2 in the second order, the sorted suspension is stained, thereby verifying whether the sorted cells are the required rare cells.

[0063] As can be seen from the above, the automated cell fractionation and loading system provided in this embodiment of the invention includes: a pump device 1, a reagent switching device 2, a sample container 3, a first solenoid valve 4, a second solenoid valve 5, a third solenoid valve 6, a weighing sensor, a bubble sensor, a controller, and a microfluidic chip 7. The pump device 1 includes an industrial syringe pump 11, a valve head 12, and a sample injector 13. The industrial syringe pump 11 is connected to the sample injector 13, and the sample outlets of the sample injector 13, the cell collection chamber 20, the waste liquid pool 30, and the microfluidic chip 7 are all connected to the valve head 12. A bubble sensor is installed on the pipeline between the sample outlet and the valve head 12. When the bubble sensor detects a bubble, it sends an early warning message to the controller. The first inlet of the microfluidic chip 7 is connected to the common pipe 50 of the reagent switching device 2 through the sample tube 40, and the second inlet of the microfluidic chip 7 is connected to the common pipe 50 through the sheath fluid tube 60. The sample container 3 is connected to the sample tube 40 through the rare cell tube 70. The sample container 3 contains a sample of rare cells with magnetic beads attached to its surface. A weighing sensor is connected to the sample container 3. The first solenoid valve 4 is located in the rare cell tube 70, the second solenoid valve 5 is located in the sample tube 40 near the common pipe 50, and the third solenoid valve 6 is located in the sheath fluid tube 60 near the common pipe 50. The controller controls the second solenoid valve 5 and the third solenoid valve 6 to open, controls the valve head 12 to connect with the sample outlet, and controls the common pipe 50 to connect sequentially with the reagent storage tanks 22 corresponding to the multiple pretreatment reagents in the reagent switching device 2 in the first order. At the same time as the common pipe 50 connects with the first reagent storage tank 22, the industrial injection pump 11 is started. When the sample is connected to the reagent storage tank 22 corresponding to the culture medium, the second solenoid valve 5 is closed and the first solenoid valve 4 is opened. The industrial syringe pump 11 draws the sample into the microfluidic chip 7 for cell sorting. When the sample is emptied based on the weight sent by the weighing sensor, the control valve head 12 switches to connect with the waste liquid tank 30. After the waste liquid is emptied, the control valve head 12 switches to connect with the sample outlet. After the sorted suspension enters the sample injector 13, the control valve head 12 switches to connect with the cell collection chamber 20, and the suspension is drawn into the cell collection chamber 20. Thus, sample loading is achieved through the pump device 1, reagent switching device 2, microfluidic chip 7, rare cell tube 70, sample tube 40, and sheath fluid tube 60, eliminating the need for pipettes, greatly reducing the amount of consumables used. The sample loading process also eliminates the need for frequent pipette tip replacement; only the reagent switching device 2 needs to be controlled. The response time is shortened from fractions of a second to milliseconds, improving efficiency. Furthermore, there is no need to reserve space for the pipetting route or pipette tip box, making the structure compact and easy to miniaturize and place.

[0064] Furthermore, the cell sorting and loading process is fully automated, requiring no human intervention.

[0065] Those skilled in the art will understand that the accompanying drawings are merely schematic diagrams of one embodiment, and the modules or processes shown in the drawings are not necessarily essential for implementing the present invention.

[0066] Those skilled in the art will understand that the modules in the apparatus of the embodiments can be distributed in the apparatus of the embodiments as described in the embodiments, or they can be located in one or more devices different from this embodiment with corresponding changes. The modules of the above embodiments can be combined into one module, or they can be further divided into multiple sub-modules.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An automated sample loading system for cell separation, characterized in that, include: The pump device includes a liquid pump, a reagent switching device, a sample container, a first solenoid valve, a second solenoid valve, a third solenoid valve, a weighing sensor, a bubble sensor, a controller, and a microfluidic chip. The liquid pump device includes an industrial syringe pump, a valve head, and a sample injector. The industrial injection pump is connected to the injector. The injector, cell collection chamber, waste liquid pool and the sample outlet of the microfluidic chip are all connected to the valve head. The pipeline between the sample outlet and the valve head is equipped with the bubble sensor. When the bubble sensor detects a bubble, it sends an early warning message to the controller. The first inlet of the microfluidic chip is connected to the common tube of the reagent switching device through a sample tube, the second inlet of the microfluidic chip is connected to the common tube through a sheath fluid tube, the sample container is connected to the sample tube through a rare cell tube, the sample container contains a sample of rare cells with magnetic beads attached to its surface, the weighing sensor is connected to the sample container, the first solenoid valve is located in the rare cell tube, the second solenoid valve is located in the sample tube near the common tube, and the third solenoid valve is located in the sheath fluid tube near the common tube; The controller controls the second and third solenoid valves to open, controls the valve head to connect with the sample outlet, and controls the common tube to connect sequentially with the reagent storage tanks corresponding to the pretreatment reagents and culture media in the reagent switching device in a first order. Simultaneously with the common tube connecting with the first reagent storage tank, the controller starts the industrial injection pump. When the common tube connects with the reagent storage tank corresponding to the culture media, the controller closes the second solenoid valve and opens the first solenoid valve. The industrial injection pump draws the sample into the microfluidic chip for cell sorting. When the sample is emptied based on the weight sent by the weighing sensor, the controller switches the valve head to connect with the waste liquid tank. After the waste liquid is emptied, the controller switches the valve head to connect with the sample outlet. After the sorted suspension enters the injector, the controller switches the valve head to connect with the cell collection chamber, and the suspension is drawn into the cell collection chamber.

2. The automated sample loading system for cell sorting as described in claim 1, characterized in that, The reagent switching device includes a multi-channel switching valve, a reagent storage tank with the same number of channels as the multi-channel switching valve, and multiple liquid level sensors respectively installed in each reagent storage tank. Each liquid level sensor detects the liquid level of the reagent in its corresponding reagent storage tank and sends the data to the controller; When the controller determines that all liquid levels are not lower than the preset liquid level threshold, it controls the second solenoid valve and the third solenoid valve to open.

3. The automated sample loading system for cell sorting as described in claim 2, characterized in that, When the controller determines that there is a liquid level below the preset liquid level threshold, it outputs an alarm message.

4. The automated sample loading system for cell sorting as described in claim 1, characterized in that, The controller determines that the weight of the sample has not changed within a preset time period based on the weight sent by the weighing sensor, controls the third solenoid valve to close, controls the valve head to switch to connect with the waste liquid pool, and after the waste liquid in the sample injector is emptied, controls the valve head to switch to connect with the sample outlet. After the sorted suspension enters the sample injector, controls the valve head to switch to connect with the cell collection chamber, and the suspension is drawn into the cell collection chamber.

5. The automated sample loading system for cell separation as described in claim 1, characterized in that, After sorting is completed, the controller controls the first solenoid valve to close, controls the second solenoid valve to open, and controls the common tube to connect sequentially with the reagent storage tanks corresponding to the multiple reagents for staining in the reagent switching device in a second order. Each reagent for staining flows sequentially into the microfluidic chip through the sample tube to stain the sorted suspension.

6. The automated cell sorting and loading system as described in claim 1, characterized in that, The sample container is connected to the sample tube near the first inlet via the rare cell tube.

7. The automated cell sorting and loading system as described in claim 1, characterized in that, The microfluidic chip includes a sample layer, a functional layer, and a magnetic field source; The sample layer includes a flow channel layer and a sealing membrane. The flow channel layer is provided with a first inlet, a second inlet and the sample outlet, and has flow channels processed inside. The sealing membrane is attached to the open surface of the flow channel layer to seal the flow channels inside the flow channel layer. The functional layer is located below the sample layer. The functional layer includes a patterned substrate, wherein a plurality of recessed slots are sequentially formed along the length direction of the patterned substrate. An amorphous alloy soft magnetic tape is embedded in each recessed slot, and the upper surface of the amorphous alloy soft magnetic tape is flush with the upper surface of the patterned substrate. The amorphous alloy soft magnetic tape faces the sealing film. The amorphous alloy soft magnetic tape is arranged sequentially along the length direction of the patterned substrate to form an array distribution parallel to the extension direction of the flow channel of the sample layer. The magnetic field source is located at the end where the sample outlet is located and on the side directly opposite the functional layer, so that a gradient magnetic field that gradually increases from the inlet to the sample outlet is formed in the flow channel for cell sorting.

8. The automated sample loading system for cell separation as described in claim 1, characterized in that, It also includes a support frame, the load-bearing surface of the weighing sensor is fixedly connected to the support frame, and the sample container is fixed to the support frame.

9. The automated sample loading system for cell sorting as described in claim 2, characterized in that, The liquid level sensor is a capacitive liquid level sensor.

10. The automated cell sorting and loading system as described in claim 1, characterized in that, The valve head is a T-type three-way valve head.

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